In this report period, we carried on our long-standing research line, development of anti-HIV drugs, however, we particularly focused our effort to develop antiretroviral agents which resist the emergence of drug-resistant HIV-1 variants, in response to the rapidly growing problem, drug failure in patients with HIV-1 infection. We also made efforts to enhance/improve antiviral efficacy in patients by physiologically altering cellular metabolisms. Over the past four years, we designed and synthesized ~300 potential HIV protease inhibitors in collaborations with intra- and extramural investigators and identified JE-2147, a novel allophenylnorstatine (apns)-containing dipeptide HIV protease inhibitor (PI) potent against a wide spectrum of HIV-1, HIV-2, SIV, and clinical HIV-1 strains, and in particular, against multi-PI-resistant HIV-1 strains with IC50 values ranging from 13-41 nM in vitro. Our structural analysis revealed that the presence of a flexible P2i moiety is important for the potency of JE-2147 toward wild type and mutant viruses. These data suggest that the use of flexible components may open a new avenue for designing PIs which resist the emergence of PI-resistant HIV-1 (Proc. Natl. Acad. Sci. USA, 96: 8675-80, 1999). It is of note that JE 2147 is currently undergoing preclinical and clinical development in the US. Most recently, we have identified another novel protease inhibitor, UIC-003, in collaboration with scientists in University of Chicago and NCI-FCRDC. This novel compound is extremely potent against HIV-1 with IC50 values of less than nanomolar concentrations (~0.5 nM). UIC-003 is also potent against multi-PI-resistant clinical strains at nanomolar concentrations (ranging from 1-6 nM). An HIV-1 population, which was propagated in vitro in the presence of increasing concentrations of UIC 003 and became less susceptible to the compound, still had IC50 values of 0.01 microM, which appear to be relatively easily maintained in vivo. Such HIV-1 mutants had a unique mutation at codon 28 of the protease-encoded gene, indicating that this novel compound binds to a unique site (or subsites) of the target protease. In 1993, we identified a set of novel mutations [Ala-62<Val(A62V), V75I, F77L, F116Y, and Q151M] in the polymerase domain of reverse transcriptase (RT), which confers on the virus a reduced sensitivity to multiple nucleo side reverse transcriptase inhibitors (NRTIs). To date, the emergence of HIV-1 resistant to multiple NRTIs (MDR) has been documented in many patient populations. Using a set of recombinant infectious clones, we also studied whether the replication kinetics of HIV-1 was altered when the virus acquired a set or subsets of the five mutations in the presence of drugs. Several MDR-HIV-1 variants were more fit than HIV-1wt in the absence of drugs emergence and that resistance-associated mutations and drug pressure are critical variants for HIV-1 fitness. We found a resistance pathway in which HIV-1MDR develops through one or more mutations, which, however, sacrifice replicative capability, then HIV-1 finally acquires optimal replication-competence by additional mutations (J. Virol. 73:5356-63, 1999). Recent X-ray analyses have shown that the role of Gln-151 of RT is critical for the formation of the 3i-pocket of RT. We have generated various recombinant HIV-1 clones possessing differing amino acid at codon 151 and virologic and enzymatic analyses are currently underway. We have recently identified two methylenecyclopropane nucleoside analogues with a phenylphosphoralaninate moiety, QYL-685 and QYL-609, which are potent against HIV-1 and HIV-2 in vitro (Antimicrob. Agents Chemother. 43:1487-90, 1999). We also induced HIV-1 variants resistant to QYL-685 in vitro which turned out to contain an M184I mutation. Viral fitness analyses revealed that the mutant had a replication disadvantage in the absence of QYL-685, while in the presence of the drug, the mutant showed greater fitness compared to HIV-1wt. These data may provide a structural and virological relevance with regard to the emergence of M184I and M184V substitutions in HIV-1 (Antimicrob. Agents Chemother. 43:2479-83, 1999). We also studied the changes of chemokine receptors on the target cells (i.e., up- or down-regulation) following viral binding or chemokine exposure and have found that upon T-cell stimulation, memory cells become more vulnerable to T-tropic HIV-1 than naive cells, while these both subsets are susceptible to macrophage tropic HIV-1. These data are related to the potential use of chemokines or chemokine receptor blockers as a new modality of HIV-1 intervention (submitted). In other area of research lines, we asked whether certain chemokine receptor expression was related to the increased risk of non-Hodgkin lymphoma (NHL) often seen in individuals with HIV-1 infection. We showed that individuals with stromal cell derived factor 1 gene variant (SDF-1-3iA) are more likely to develop NHL (Blood 93:1838-42, 1999) and have more recently found that individuals who develop NHL have significantly higher levels of SDF-1 mRNA, preceding at least 1 to 2 years prior to the diagnosis, as compared to individuals without NHL (p < 0.0001). The data strongly suggest that concurrent up-regulation of SDF and the higher numbers of integrated HIV-1 DNA copies increase the risk of AIDS-related lymphoma, which may have a clinical relevance (submitted).